1. Field of the Invention
There is a continuing and increasing need for accurate, sensitive techniques for measuring trace amounts of organic materials in a wide variety of samples. This need includes the measurement of drugs, naturally occurring physiological active compounds or nutrients in physiological fluids, the presence of trace amounts of contaminants or toxic materials in foods, water or other fluids, and the like, as well as monitoring materials for trace contamination introduced during chemical processing.
Among the various techniques which have found increasing exploitation are techniques involving receptors which recognize or bind to a specific polar and spatial organization of one or more molecules. For the most part, the receptors are antibodies and the techniques are referred to as immunoassays. These techniques conventionally employ a labelled ligand, where the binding to the receptor allows for distinguishing between a bound labelled ligand and an unbound labelled ligand. Certain techniques, generally referred to as heterogeneous, rely on segregating the bound from the unbound labelled ligand. Other techniques, generally referred as homogeneous, rely on the bound labelled ligand providing a signal level different from unbound labelled ligand.
Methods are known for the detection of polyvalent antigens wherein at least two different binding sites on the antigen are used. Two-site immunometric assays are well known. They are used to detect the presence or concentration of a multideterminant antigen in a liquid sample. They involve reacting the antigen with both an immobilized antibody directed against one of the antigenic determinants and an antibody that is directed against another of the antigenic determinants and is indirectly or directly labelled to permit detection of the resulting immune complex. It is also known to use polyclonal antibodies, monoclonal antibodies, or a combination of both polyclonal and monoclonal antibodies. The use of two antibodies in the immunoassay enhances the sensitivity of the assay by permitting the use of excess antibody to ensure complete binding of antibody to all of the analyte in the sample. The use of monoclonal antibodies in a two-site immunoassay in some cases also increases the specificity of the assay by requiring the presence of two epitopic sites on the same molecule to produce a positive result.
While such assays involving two antibodies are useful where the analyte is a polyvalent antigen, the benefits realized by employing two antibodies are not available for monoepitopic antigens such as drugs or other organic compounds.
2. Description of the Related Art
The use of double antibodies for enhanced sensitivity in immunoassays is described in U.S. Pat. No. 4,281,061. Two-site immunoassays using monoclonal antibodies of different classes or subclasses and test kits for performing such assays are disclosed in U.S. Pat. No. 4,474,892. A method for the detection and/or determination of a polyvalent antigen using at least two different monoclonal antibodies is described in U.S. Pat. No. 4, 471,058. U.S. Pat. No. 4,486,530 discloses immunometric assays using monoclonal antibodies. Monoclonal antibody mixtures and their use for enhanced sensitivity in immunoassays is discussed in U.S. Pat. No. 4,514,505. The use of anti-idiotype antibodies in immunoassays is discussed in U.S. Pat. No. 4,536,479. U.S. Pat. No. 4,062,935 discloses an immunoassay involving the binding of RF to the antigen-antibody complex. Sensitive immunoassays of antigens or antibodies sequestered within immune complexes is disclosed in U.S. Pat. No. 4,459,359. An assay of immune complexes is discussed in U.S. Pat. No. 4,141,965. U.S. Pat. No. 4,420,461 discloses aggulination-inhibition test kits for detecting immune complexes. An enhancing antibody being a novel component of the immune response is described by Nemazee, et al., Proc. Natl. Acad. Sci., USA, 79:3828-3832 (1982).